This invention claims priority of a German filed patent application 199 44 148.0 which is incorporated by reference herein.
The invention concerns a laser scanning microscope, preferably a confocal laser scanning microscope, having an illumination beam path extending between a laser light source and a specimen, and a detection beam path extending between the specimen and a detection device.
Laser scanning microscopes of the generic type have been known for some time and are used, among other applications, in the semiconductor industry for wafer inspection and in biomedical basic research. Reference is made, purely by way of example, to the German Patent Application DE-A-43 30 347. DE-A-43 30 347 discloses a generic laser scanning microscope which is suitable in particular for the biomedical application in which the specimen being examined is specifically labeled with various fluorescent dyes. After excitation with suitable laser light sources, the fluorescent light of the fluorescent dyes can be detected.
Laser scanning microscopes of the generic type have so far been used only to perform relative measurements. In this context, it is possible only for one specimen at a time to acquire fluorescent light from the fluorescent dye distribution present in the specimen and then, in the case where various fluorescent dyes are being detected, to correlate them quantitatively with one another with sufficient accuracy. When a further specimen is measured with the same laser scanning microscope, it would be desirable to correlate the measured image data of these two specimens with one another with corresponding accuracy. Quantitative comparative measurements on different specimens would be necessary principally for diagnostic applications in the medical field. This has heretofore not been possible because in laser scanning microscopes of the generic type, no apparatus for calibrating the relevant assemblies in the microscope is provided, and furthermore the individual assemblies of a laser scanning microscope are subject to short-term and long-term fluctuations due to external influences, which make comparative measurements of different specimens impossible even if the microscope assemblies are suitably calibrated with sufficient accuracy.
Sufficient calibration of the relevant assemblies of a laser scanning microscope is possible, for example, with the apparatus known from the German Patent Application DE-A-199 06 763, although the short-term and long-term fluctuations due to external influences cannot be compensated for therein. These influences include principally the temperature dependences of individual assemblies of the microscope, for example of the detector or the filters that are used, as well as laser intensity fluctuations (mode hopping).
It is therefore the object of the present invention to describe a laser scanning microscope, preferably a confocal laser scanning microscope, with which it is possible to perform absolute measurements on different specimens with sufficient accuracy, and in that context to correct for disruptive influences on the measurement conditions. Another object of the invention is to describe a method for achieving the aforesaid object.
The aforementioned object is achieved by a laser scanning microscope, preferably a confocal laser scanning microscope for inspecting a specimen, comprises a laser light source defining an illumination beam path extending between the laser light source and the specimen, a detection device a defining a detection beam path extending between the specimen and the detection device, at least one reference beam path for reference measurement wherein reference light is coupled out of the illumination beam path into the reference beam path, and the reference light is qualitatively and quantitatively detectable by the detection device.
Furthermore the above object is as well achieved by a method for reference correction in a laser scanning microscope, preferably a confocal laser scanning microscope having an illumination beam path extending between at least one laser light source and a specimen, and a detection beam path extending between the specimen and at least one detection device, comprises the steps of:
coupling reference light out of the illumination beam path (4) into a reference beam path (9);
using the reference light for error compensation; and
detecting the reference light qualitatively and quantitatively with a detection device (6).
What has been recognized first of all according to the present invention is that with conventional laser scanning microscopes, comparative measurements of different specimens are not possible even when suitable calibration devices with an absolute accuracy are used, due to short-term and long-term fluctuations in individual assemblies of the laser scanning microscope. It has been recognized that above all intensity fluctuations in the laser (e.g. mode hopping), as well as changes in the temperature of the filters and of the detector, can negatively influence or falsify the comparative measurements of different specimens.
According to the present invention, therefore, the laser scanning microscope has a reference beam path with which it is possible to perform corresponding reference measurements. In this context, reference light is coupled out of the illumination beam path into the reference beam path and is detected by a detection device. As a result, in addition to the actual measurement of the specimen, reference measurements are performed with which the short-term and long-term fluctuations of the individual components of the laser scanning microscope can be compensated for.
The reference beam path extends between the coupling-out point and the detection device, the coupling-out point being provided in the illumination beam path of the laser scanning microscope. The laser light of the laser light source coupled into the reference beam path will hereinafter be called xe2x80x9creference light.xe2x80x9d
In terms of a concrete embodiment, the coupling-out point is located between the laser light source and the beam splitter device. This ensures that only light from the laser light source is coupled into the reference beam path, i.e., for example, no specimen fluorescent light components or conventional microscope transmitted light components are superimposed on the reference light.
If an acousto-optical beam splitter (AOBS) is used as the beam splitter device, it is advantageous if the coupling-out point is located directly at the AOBS. A laser scanning microscope having an AOBS is described in the German Patent Application DE-A-199 06 757.0.
Advantageously, further optical components are arranged in the reference beam path. These include a diffusion disk that comprises, for example, a glass plate with a roughened surface or a glass plate made of milk glass. Also arranged in the reference beam path is at least one beam deflection element, so that beam guidance of the reference beam can thereby be implemented.
In terms of a concrete embodiment, at least one light-sensitive sensor with which reference light can be detected is arranged in the reference beam path. This light-sensitive sensor is preferably provided only for a partial beam of the reference beam.
A beam attenuator (gray filter or neutral glass filter), with which the reference light intensity can be adapted to the properties of the detection unit, is arranged in the reference beam path. Advantageously, the transmissivity of the beam attenuator is selected such that the reference light intensity is of the same order of magnitude as the light intensity to be detected from the specimen, so that approximately the same dynamic range is present for detection of the reference light and the detected light.
A filter receiving element is also arranged in the reference beam path and acts on the illumination beam path and/or detection beam path. This can be a conventional filter wheel or a linearly arranged filter slider, into which or more different filters can be inserted. Coated glass plates that have wavelength-specific reflection or transmission characteristics are usually used as filter inserts. The use of colored glass plates or so-called holographic notch filters is also quite common. The filter receiving element is arranged movably, i.e. for example rotatably or displaceably in a linear direction. With the filter receiving element fitted with filter inserts, it is possible to select the beam to be detected using the detection device. Advantageously, for example, when the filter receiving element is in one position only light from the detection beam is selected for the detection device, when the filter receiving element is in another position only light from the reference beam is selected for the detection device, and when the filter receiving element is in a further position, no light is selected for the detection device in order to measure a dark current.
Since the filters used in the filter receiving element are in some cases temperature-dependent, and any temperature change results in a change in the transmission characteristic of the filters, the filter receiving element is kept at a constant temperature. For that purpose, the entire filter receiving element is heated with the aid of a suitably controlled heating system, in particular with a heating resistor.
In a preferred embodiment, the further optical elements in the reference beam path are arranged after the diffusion disk, as viewed outward from the coupling-out point. The coherent laser light coupled from the laser light source into the reference beam path is no longer coherent after passing through the diffusion disk, so that the interaction between the light passing through the diffusion disk and the further optical elements in the reference beam path does not cause any undesirable interference phenomena. Interference phenomena of this kind have an extremely disruptive effect on measurements, since even the slightest changes in temperature, or changes in path length thereby induced, in the optical beam path are associated with an intensity modulation of the light being measured.
Advantageously, a thick beam splitter, which can be configured as a plane-parallel glass plate, is used as the coupling-out element. In a preferred embodiment, this beam splitter is at least four millimeters thick and is arranged at a 45xc2x0 angle to the illumination beam path, and at least one further light beam in addition to the reference beam is coupled out. The further light beam could be, for example, the secondary reflection at the beam splitter plate of the laser light coming from the laser light source. The xe2x80x9csecondary reflectionxe2x80x9d is understood to be the first internal reflection occurring in the thick beam splitter plate at the glass surface facing away from the laser light source, which emerges in the direction of the reference beam path after passing through the glass surface of the beam splitter plate that faces toward the laser light source. What therefore results, as a function of the thickness of the beam splitter plate used, is a lateral parallel offset of the additionally coupled-out light beam with respect to the reference beam.
The intensity of the additionally coupled-out light beam is detected or measured with a light-sensitive sensor. The signal thus measured, which is directly proportional to the output power of the laser light source used, is used as the reference signal for the detection device. In a preferred embodiment, the reference signal thus obtained is used as the reference voltage for the digitization device. It is thus possible, advantageously and with simple means (only one corresponding photodiode), to correct the intensity fluctuation of the laser light source that negatively affects the specimen measurement. In particular, it is possible thereby to correct short-term intensity fluctuations of the laser light source (such as mode hopping), if that correction is, advantageously, performed during the recording of data from the specimen, i.e. the actual measurement operation.
With regard to the correction of, in particular, long-term fluctuations in the components built into the microscope, it is advantageous to correct the image data measured with the laser scanning microscope suitably using the reference signals measured with the reference beam. The result of this action is principally to counteract the long-term drift of the detection device or the laser light source. This drift causes an offset within the measured image data, which is precisely the reason why a quantitative comparison of measurements at different points in time cannot be made with the requisite absolute accuracy. Concretely, the correction of the measured image data in the digitization device could be accomplished with the aid of an input lookup table. This input lookup table (LUT) can be implemented in such a way that the intensities of the measured image data during the digitizing step are digitized in accordance with the measured reference signals of the reference beam in such a way that a further correction step for the image data digitized in that manner is no longer necessary. The advantage of this procedure is that the correction can be performed xe2x80x9conline,xe2x80x9d i.e. already during image recording.
With regard to a further embodiment, at least one further light source, which for example can be used for purposes of calibrating the optical components present in the reference beam path, could be provided in the reference beam path. It is also conceivable for at least one further detector to be arranged in the reference beam path. For example, the light intensity in the reference beam path upstream from an optical element of the reference beam path could be measured with an additional detector, and the intensity downstream from that optical element could be measured with an additional detector, so as thereby to be able to eliminate or compensate for possible interference effects on that optical element.
In order to compensate for the temperature dependence of the optical elements that are arranged in the illumination beam path and/or detection beam path but not in the reference beam path, those elements could be kept at a constant temperature. This could again be accomplished, as in the case of the filter receiving element, with a controlled heating system.
Short-term and long-term fluctuations of the individual components of the laser scanning microscope can thereby advantageously be compensated for; in particular, by way of the reference measurement occurring simultaneously with the specimen measurement it is possible to compensate for short-term fluctuations of the components.
With regard to compensation for long-term fluctuations of the assemblies present in the laser scanning microscope, provision is advantageously made for measuring, directly before and/or after each measurement cycle, reference signals with which the measured specimen image data can be corrected. Quantitative values such as the instantaneous laser light output of the light source used, or the dark current of the detection device, may be of particular interest in this context. A measurement cycle could comprise a one-, two-, or three-dimensional data recording.
For the reference correction, at least the measured variables listed below are measured, specifically the dark current I0, reference R, and dark current R0 of the reference.
The measured image data IXYZ are corrected in suitable fashion using the measured reference signals, preferably according to the equation:
IXYZ,KORR=F*(IXYZxe2x88x92I0)/(Rxe2x88x92R0),
in which what is used for the variable F is a suitable scaling factor that ultimately maps the measured image data, during correction, onto a suitable digitization region, for example onto the 12-bit region comprising the gray values from 0 to 4095.
To simplify the reference correction, I0=R0 could be set, specifically if, on the basis of the measurements made with the reference correction that is actually used, there is little difference in value between the dark current R0 of the reference and the dark current I0 of the system when the light source is blanked out.